1. Field of the Invention
The present disclosure relates to novel nucleic acid ligands (aptamers) that bind to the Listeria outer membrane protein targets internalin A (InlA; Lmo0433), internalin E (InlE; Lmo0264), and Lmo0610. The disclosed aptamer reagents can be used to screen samples such as food, clinical, and environmental samples for the presence of InlA, InlE, and Lmo0610. The novel DNA aptamers can also potentially be used in various applications in which the presence or absence of Listeria is required.
2. Description of the Related Art
An estimated 76 million foodborne illnesses occur each year in the United States, resulting in 325,000 hospitalizations and 5000 deaths (see Mead et al., “Food-Related Illness and Death in the United States,” Emerg. Infec. Dis. 5, 607-25 (1999)). Listeria monocytogenes has been implicated in at least 11 human foodborne epidemics worldwide and is associated with foods that are ready-to-eat and can be consumed without cooking (see Ben Embarek, P. K., “Presence, Detection and Growth of Listeria Monocytogenes in Seafoods: a review,” Int. J. Food Microbiol. 23, 17-34 (1994)). Although Listeria monocytogenes causes only 2500 cases of foodborne illness per year, it is responsible for 10% of the total foodborne illness-related deaths. The majority of human listeriosis cases occur in neonates, the elderly, and immuno-compromised individuals, with case fatality rates of 20-40% (see Farber et al., “Listeria monocytogenes, a Food-Borne Pathogen,” Microbio. Rev. 55, 476-511 (1991); Schuchat et al., “Epidemiology of Human Listeriosis,” Clin. Microbiol. Rev. 4, 169-83 (1991); “Update—Multistate Outbreak of Listeriosis,” Centers for Disease Control & Prevention Morbid. Mortal. Weekly Rep. 47, 1117-18 (1999); Jacquet et al., “Investigations related to the epidemic strain involved in the French Listeriosis outbreak in 1992,” Appl. Environ. Microbiol. 61, 2242-46 (1995)). Because of the severity of the illness and its association with foods that can be consumed without heating, the U.S. Food and Drug Administration (FDA) and Food Safety and Inspection Service (FSIS) established a zero tolerance policy for the presence of Listeria monocytogenes in ready-to-eat (RTE) foods in 1989.
The increasing number of governmental regulations and the changing topography of food processing and manufacturing have spurred the development of faster, more sensitive, and cost-effective technologies for pathogen detection. Currently, there are many different methods available for Listeria monocytogenes and Listeria spp. detection on the market. The most widely used method, due to cost and sensitivity considerations, is the traditional microbiological method of plating. Although the currently available methods are effective for recovery of Listeria monocytogenes from a variety of samples, positive results are not obtained until 5-7 days after sample collection. Rapid methods that employ nucleic acid amplification and immunochemical techniques reduce the time needed to obtain results compared to culture-based methods and offer the possibility of high throughput automation. The rapid methods currently on the market comprise PCR, probe hybridization, enzyme-linked immunoassay (ELISA), enzyme-linked fluorescent assay (ELFA), lateral flow, and magnetic bead-based methods. The time needed to obtain results decreases to 2-4 days for these assays, but most require enrichment steps to improve sensitivity and allow recovery of injured or stressed organisms.
The faster time-to-results and high throughput capabilities have led to increased adoption of PCR methods in food testing, but the greater costs associated with use of PCR methods as compared to traditional culture methods and their lack of universal acceptance currently restricts the widespread use of molecular methods in general. PCR-based methods also have several limitations. Theoretically, PCR-based technology should provide the detection level of ≦1 CFU/25 g food sample mandated by the zero tolerance regulation. Assay sensitivity, however, is complicated by a number of factors, including low contamination levels, large sample volumes relative to reaction volumes, and inhibition of the PCR reaction by components of the food matrix. Thus assay sensitivities typically do not reach theoretical values (see Norton, D. M., J. AOAC Int. 85, 505-15 (2002)). Also, PCR only detects the presence of DNA and cannot indicate whether the pathogens are dead or alive.
By contrast, immunological methods rely on the interaction between specific antibodies to selectively capture, label, or detect a target organism and are widely used and accepted for the detection and confirmation of specific microorganisms. The widespread use and acceptance of immunology-based methods has resulted in a vast array of commercial test kits for the detection of the most common foodborne bacteria in foods, including Salmonella, Listeria, Campylobacter, and E. coli O157:H7. ELISAs, which are the most common format used for immunological detection, have detection limits of between 103-105 cfu/mL (see Churchill et al., “Detection of Listeria monocytogenes and the toxin listeriolysin O in food,” J. Microbiol. Meth. 64, 141-70 (2006)). To achieve this detection limit often requires enrichment of the pathogens for at least 24 hours before the sample is adequate for detection by ELISA (see de Boer et al., “Methodology for detection and typing of food borne microorganisms,” Int. J. Food Microbiol. 50, 119-30 (1999)).
Despite the improved time-to-results of many rapid detection systems, the requirement of conventional cultural enrichment still remains an important limiting feature of these methods. Also, these methods lack the ability to detect biomolecules in real time. There is an increasing demand for simple, inexpensive, and reliable tests to analyze food samples. Biosensor technology has the potential to meet these needs in or near real time (see Alocilja et al., “Market analysis of biosensors for food safety,” Biosensors & Bioelectronics 18, 841-46 (2003); Hall, “Biosensor technologies for detecting microbiological food borne hazards,” Microbes & Infection 4, 425-32 (2002); Deisingh et al., “Biosensors for the detection of bacteria,” Can. J. Microbiol. 50, 69-77 (2004)). Studies have shown that biosensors can detect a broad spectrum of analytes in complex samples with minimal sample pre-treatment (see Hall, “Biosensor technologies for detecting microbiological food borne hazards,” Microbes & Infection 4, 425-432 (2002); Deisingh et al., “Biosensors for the detection of bacteria,” Can. J. Microbiol. 50, 69-77 (2004)).
Biosensors for bacterial detection generally involve a biological recognition component such as receptors, nucleic acids, or antibodies in contact with physical or chemical transducers. Depending on the method of signal transduction, biosensors can be divided into five basic types: electrochemical, optical, piezoelectric, thermal, and magnetic. Recently, sensors have been developed for detection of Listeria monocytogenes (see Geng et al., “Detection of Low Levels of Listeria monocytogenes Cells by Using a Fiber-Optic Immunosensor,” Applied & Environmental Microbiology 70, 6138-46 (2004); Leonard, P. et al., J. Food Prot. 68, 728-35 (2005); Leonard et al., “A generic approach for the detection of whole Listeria monocytogenes cells in contaminated samples using surface Plasmon resonance,” Biosensors & Bioelectronics 19, 1331-35 (2004); Tims, T. B. et al., “Detection of low levels of Listeria monocytogenes within 20 hours using an evanescent wave biosensor,” Am. Clin. Lab. 20, 28-29 (2001)). The sensitivity and specificity of these assays are dependent on the specific antibody that is used for detection. The sensitivity threshold for a fiber-optic immunosensor (Analyte 2000; Research International, Woodinville, Wash.) was measured to be approximately 103 CFU/mL for a pure culture of Listeria monocytogenes and 104 CFU/mL when grown with lactic acid bacteria (Geng et al., “Detection of Low Levels of Listeria monocytogenes Cells by Using a Fiber-Optic Immunosensor,” Applied & Environmental Microbiology 70, 6138-46 (2004)). These levels of detection compare with immunological methods, as expected, since antibodies were the capture agents in contact with the transducer. Both polyclonal and monoclonal antibodies have been used for biosensor studies. Polyclonal antibodies have been used as detection reagents for several decades (see Breitling, F., Dubel, S. Recombinant Antibodies 154 (John Wiley & Sons Inc. 1999)). The supply of polyclonal antibodies is limited and repeated immunizations are required to replenish depleted stocks. By contrast, monoclonal antibodies offer a continuous supply of homogeneous, well-characterized antibodies. High cost, low yields, and the requirement of skilled labor are some of the problems associated with monoclonal antibody production.
Aptamers, first reported in 1990 (see Tuerk, C., Gold, L., Science 249, 505-10 (1990); Ellington et al., “In vitro selection of RNA molecules that bind specific ligands,” Nature 346, 818-22 (1990)), offer ideal candidates for use as the biological recognition components in biosensors, possessing advantages over traditional antibodies for use in sensors (see Jayasena, “Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics,” Clin. Chem. 45, 1628-50 (1990)). Aptamers are nucleic acid ligands that can be generated against amino acids, drugs, proteins, and complex targets such as cells (see Gopinath, S. C. et al., “An RNA aptamer that distinguishes between closely related human influenza viruses and inhibits hemagglutanin-mediated membrane fusion,” J. Gen. Virol. 87, 479-487 (2006); Cerchia, L. et al., “Neutralizing Aptamers from Whole-Cell SELEX Inhibit the RET Receptor Tyrosine Kinase,” PLoS Biol. 3, e123 (2005); Duconge, F. et al., PLos Biol. 3, e123 (2005); Mori, T. et al., “RNA aptamers selected against the receptor activator of NF-kB acquire general affinity to proteins of the tumor necrosis factor receptor family,” Nuc. Acids Res. 32, 6120-28 (2004); Daniels, D. A. et al., “A tenascin-C aptamer identified by tumor cell SELEX: Systematic evolution of ligands by exponential enrichment,” Proc. Natl. Acad. Sci. 100, 15416-21 (2003)). Numerous aptamers have been selected using this technique against a wide range of targets, with selectivity, specificity, and affinity equal and sometimes superior to those of antibodies. The technique in which these oligonucleotide ligands are obtained was termed SELEX (Systematic Evolution of Ligands by Exponential Enrichment), described in U.S. Pat. Nos. 5,475,096 and 5,270,163. The advantages of using aptamers over traditional antibodies for in vitro assays include: 1) the ability to be denatured/renatured multiple times (reusable), 2) stability in long term storage and the ability to be transported at ambient temperature, 3) the ability to adjust selection conditions to obtain aptamers with properties desirable for in vitro assay, 4) generation by chemical synthesis, resulting in little batch to batch variation, 5) selection through an in vitro process eliminating the use of animals, and 6) the ability to attach reporter molecules at precise locations (see O'Sullivan, C. K., “Aptasensors—the future of biosensing?” Anal. Bioanal. Chem. 372, 44-48 (2002)).
Aptamers have yet to be used in diagnostic or biosensor approaches for food-borne pathogen detection. The aptamers isolated against outer membrane proteins in Listeria may be used in diagnostic and biosensor detection technologies for food, clinical, or environmental samples.